Sustainable and renewable sources of liquid fuel to operate machinery, aircraft, and vehicles are necessary to reduce the amount of carbon dioxide emissions in the atmosphere, as well as to reduce global energy consumption based upon coal, oil, and natural gas economies.
Increased demand for energy by the global economy has placed increasing pressure on the cost of hydrocarbons. Aside from energy, many industries, including plastics and chemical manufacturers, rely heavily on the availability of fossil hydrocarbon sources as a feedstock for their manufacturing processes. Cost-effective alternatives to current sources of supply could help mitigate the upward pressure on fossil resource demand and raw material costs.
Biologic systems that fix carbon through natural biochemical metabolic processes are known. Algal systems have been developed to create hydrocarbons through photosynthetic reactions, as well as heterotrophic reactions fed by sugar that indirectly depend upon photosynthesis, but insufficient yields limit the effectiveness, economic feasibility, practicality and commercial adoption. Bacterial cells have been genetically engineered to process sugar feedstocks into useful hydrocarbons in heterotrophic fermentation systems, however, there are significant drawbacks for these systems.
Heterotrophic fermentations are vulnerable to contamination because heterotrophic microorganisms that can grow on fixed carbon nutrients are far more ubiquitous in the surface environment. Heterotrophic technologies also generally suffer limitations in terms of food versus fuel conflict and negative environmental impacts.
Gas-to-liquid (GTL) technologies have the benefit of allowing the utilization of waste carbon sources—including highly lignocellulosic waste through the conversion to synthesis gas (syngas) via gasification, as well as waste CO2 through the provision of reduced hydrogen—in the production of liquid fuels and/or organic chemicals. Syngas is a mix of gases that generally contains H2, CO, and CO2 as major components, which can be generated through steam reforming of methane and/or liquid petroleum gas or through gasification of any organic material, including but not limited to biomass, waste organic matter, various polymers, and coal. Many gasification processes are available for the production of syngas. A number of gasification processes subject the carbonaceous feedstock to partial oxidation at high temperatures (500-1500° C.), with the oxygen supply restricted to prevent complete combustion, producing syngas with varying composition depending on feedstock and reaction conditions such that the ratio of H2:CO can range from 0.5:1 to 3:1. The hydrogen component of syngas can be raised through the reaction of CO with steam in the water gas shift reaction with a concomitant increase in CO2 in the syngas mix.
Some major technologies for syngas conversion to liquid fuels or chemicals include chemical catalytic processes such as the Fischer-Tropsch (F-T) as well as processes for the synthesis of methanol or other mixed alcohols, and biological gas fermentation processes. F-T has been worked on for almost one hundred years and relies on metal-based, inorganic catalysts for the conversion of syngas into longer chain hydrocarbons. Difficulties with F-T include: a wide chain length distribution of products resulting in the need to reprocess short chain length products such as methane and LPG and/or the need to perform additional costly post-processing steps on long chain waxes and tars such as hydrocracking; high catalyst sensitivity to syngas impurities such as sulfur containing compounds, tars, and particulates, generally necessitating multiple costly gas clean up steps; relatively low flexibility in terms of accommodating various ratios of syngas constituents i.e. H2:CO, and low tolerance of CO2, often resulting in additional costly syngas conditioning steps such as water gas shift and CO2 removal; the actual F-T step is relatively high temperature and pressure resulting in costly compression and heating requirements; the wide distribution of products generally necessitates the storage, handling, and transport of a wide array of products which is often uneconomic except for relatively large scale operations; F-T products (e.g. diesel, jet fuel, naphtha, waxes) are relatively low in value at current (2011) prices compared to many different higher value oils, lipids, and oleochemicals that can be produced biologically. The difficulties with F-T generally also apply to other chemical conversion processes such as methanol synthesis.
The gasification of biomass to generate syngas has a long history going back to World War II where biomass gasification was used for running modified automobiles, boats, buses, and trucks. Presently, a number of biomass gasification technologies are at, or near commercialization (able to gasify 10,000 or more tons of biomass per year), and are generally used for the production of heat and/or electricity. The synthesis of chemicals or fuels from syngas generated via biomass gasification is at an earlier stage of development, and is generally pre-commercial.
Using syngas and/or CO2 and/or renewable H2 in gas fermentation enables the utilization of cheaper and more flexible sources of energy and/or carbon for the biological synthesis of sustainable chemicals and fuels than is possible through heterotrophic or phototrophic synthesis. In gas fermentation, syngas acts as both a carbon and energy source for the microbial culture. Some of the advantages of syngas fermentation include: the production of a relatively narrow range of carbon chain length distribution compared to F-T; lower sensitivity to syngas impurities; greater tolerance of varying ratios of H2:CO and the presence of CO2; able to operate at much closer to ambient temperature and pressure; able to produce various higher value oleochemical products.
A fermentation process based upon a gaseous feedstock such as syngas can allow for far lower negative environmental and food production impacts in the biological synthesis of liquid fuels and/or chemicals than the highly land and water intensive heterotrophic or phototrophic-based technologies. However, current biological GTL technologies generally yield relatively short chain alcohols, or other short chain organic compounds, as products, which have relatively low energy density and infrastructure compatibility with current petrochemical and oleochemical processes.
The syngas-growing microorganisms used in current biological GTL technologies are generally inappropriate for the synthesis of high energy density, infrastructure compatible fuels, or other longer carbon chain lipid-based chemicals. Their short chain products are relatively low in value and they generally don't efficiently synthesize drop-in fuels such as diesel or jet fuel, or higher value lipid-based chemicals.
Furthermore the types of microorganisms used in current biological GTL technologies such as Clostridia have a relatively low tolerance for their short carbon chain gas fermentation products such as ethanol, butanol, or acetic acid, which limits titers and complicates product recovery, hurting the overall economics of the GTL process.
There is a need to identify a set of microorganisms that can grow in conventional and scalable contained reaction vessels and that produce commercially viable sets of organic carbon chains of at least eight carbon atoms long in a commercially feasible method. There is a need to identify microorganisms not limited metabolically by typically used carbon and energy inputs such as sugars, and a microorganism that can additionally utilize syngas, producer gas, as well as a wide array of abiotic sources of carbon and energy for the synthesis of drop-in fuels and chemicals, leading to a feedstock flexibility for the present technology that far exceeds comparable heterotrophic systems. There is a need to identify and use microorganisms that can utilize electron donors such as hydrogen, present in syngas, producer gas, as well as readily generated through a wide array of abiotic renewable energy technologies, for growth and carbon fixation.
The targeting of fatty acids produced through fatty acid biosynthesis to relatively shorter fatty acid chain lengths from C8-C14 has been achieved in heterotrophic microorganisms. This has been accomplished through the use of thioesterases to change populations of fatty acids C8-C14 and the over-expression of thioesterases to increase shorter chain length fatty acids. Examples in the prior art include C8-C14 thioesterase expression to produce shorter chain lengths in U.S. Pat. No. 7,883,882 Renewable chemical production from novel fatty acid feedstocks, Franklin et al. Solazyme, p. 58.
However there is a need to target the production of shorter chain length fatty acids in microorganisms that are capable of growing and producing lipids chemotrophically on syngas or H2/CO2 gas mixes to enable microbial GTL production of lipids with targeted, mid-length carbon chains.
Dicarboxylic acids (Diacids) such as dodecanedoic acid (n=10) are used in production of nylon (nylon-6,12), polyamides, coatings, adhesives, greases, polyesters, dyes, detergents, flame retardants and fragrances. Diacids can be produced by fermentation of long-chain alkanes by candida tropicalis (Kroha K, Infom 2004, 15, 568). Traumatic acid, monounsaturated dodecanedoic acid (10E-dodeca-1,12-dicarboxylic acid) has been produced from plant tissues English J et al., Science 1939, 90, 329. Pyrococcus furiosus produces an array of dicarboxylic acids (Carballeira, 1997). The total amount of dicarboxylic acids comprises only 3.4% of the total, however, this could be boosted by various literature methods.
There is a need for a biological, non-heterotrophic means of producing diacids from low-cost or sustainable syngas feedstocks.
Nutritionally important n-3 fatty acids include α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), all of which are polyunsaturated. N-3 fatty acids that are important in human physiology are α-linolenic acid (18:3, n-3; ALA), eicosapentaenoic acid (20:5, n-3; EPA), and docosahexaenoic acid (22:6, n-3; DHA). These three polyunsaturates have either 3, 5, or 6 double bonds in a carbon chain of 18, 20, or 22 carbon atoms, respectively. As with most naturally produced fatty acids, all double bonds are in the cis-configuration.
A fatty acid desaturase is an enzyme that removes two hydrogen atoms from a fatty acid, creating a carbon/carbon double bond. These desaturases are classified as delta—indicating that the double bond is created at a fixed position from the carboxyl group of a fatty acid (for example, Δ9 desaturase creates a double bond at the 9th position from the carboxyl end). omega (e.g. ω3desaturase)—indicating the double bond is created between the third and fourth carbon from the methyl end of the fatty acid. In the biosynthesis of essential fatty acids, an elongase alternates with different desaturases (for example, Δ6desaturase) repeatedly inserting an ethyl group, then forming a double bond.
Most polyunsaturated oils come from fish and there is a need for alternate, and particularly microbial sources of polyunsaturated fatty acids, given depleting fish stocks and increasing pollution in the oceans.